Isothermal nucleic acid amplification

ABSTRACT

The present disclosure relates to methods and compositions for ATP regeneration system for use in conjunction with the strand invasion step of an isothermal nucleic acid amplification which uses recombinases that utilize ATP as an energy source.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/709,793, which was filed on May 12, 2015, now issued as U.S. Pat. No.9,657,340, which is a divisional of U.S. patent application Ser. No.12/997,382, which was filed on Dec. 10, 2010, as a National Phase filingunder 35 U.S.C. § 371 of International Patent Application No.PCT/GB2009/050662 filed on Jun. 11, 2009, which claims priority to: (1)GB patent application no. 0810650.2 filed on Jun. 11, 2008; and (2) GBpatent application no. 0822533.6 filed on Dec. 11, 2008. The entiredisclosures of each of the prior applications are hereby incorporatedherein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a sequence listing in computer readable form;the file, 3073134B_seqlisting.txt is 7.5 KB in size. The file is herebyincorporated into the instant disclosure.

FIELD OF THE INVENTION

The invention relates to the amplification of nucleic acids, inparticular to an isothermal process for amplifying a double-strandednucleic acid target molecule.

BACKGROUND OF THE INVENTION

Within nucleic acid and genetic material technologies, it is oftennecessary to determine whether a gene, a part of a gene, or a nucleotidesequence is present in a living organism, a cellular extract of thisorganism, or a biological sample. Since any gene or part of a gene ischaracterized by a specific sequence of nucleotide bases, it is onlynecessary to search directly for the presence of all or part of saidspecific sequence in a sample containing a mixture of polynucleotides.

There is enormous interest in this search for specific polynucleotidesequences, particularly in detection of pathogenic organisms,determination of the presence of alleles, detection of the presence oflesions in a host genome, or detection of the presence of a particularRNA or modification of a cell host. Genetic diseases such asHuntington's disease, Duchenne's disease, phenylketonuria, and betathalassemia can thus be diagnosed by analyzing nucleic acids from theindividual. Also it is possible to diagnose or identify viruses,viroids, bacteria, fungi, protozoans, or any other form of plant oranimal life by tests employing nucleic probes.

Once the specific sequence of an organism or a disease is known, thenucleic acids should be extracted from a sample and a determinationshould be made as to whether this sequence is present. Various methodsof nucleic acid detection have been described in the literature. Thesemethods are based on the properties of purine-pyrimidine pairing ofcomplementary nucleic acid strands in DNA-DNA, DNA-RNA, and RNA-RNAduplexes.

This pairing process is effected by establishing hydrogen bonds betweenthe adenine-thymine (A-T) and guanine-cytosine (G-C) bases ofdouble-stranded DNA; adenine-uracil (A-U) base pairs can also form byhydrogen bonding in DNA-RNA or RNA-RNA duplexes. The pairing of nucleicacid strands for determining the presence or absence of a given nucleicacid molecule is commonly called “nucleic acid hybridization” or simply“hybridization”.

The most direct method for detecting the presence of a target sequencein a nucleic acid sample is to obtain a “probe” whose sequence issufficiently complementary to part of the target nucleic acid tohybridize therewith. A pre-synthesised probe can be applied in a samplecontaining nucleic acids. If the target sequence is present, the probewill form a hybridization product with the target. In the absence of atarget sequence, no hybridization product will form. Probe hybridizationmay be detected by numerous methods known in the art. Commonly the probemay be conjugated to a detectable marker. Fluorescent or enzymaticmarkers form the basis of molecular beacons, Taqman and other cleavableprobes in homogeneous systems. Alternatively the probe may be used tocapture amplified material or labelled such that the amplicon isdetected after separating a probe hybridized to the amplicon fromnon-hybridized material.

The main difficulty in this approach, however, is that it is notdirectly applicable to cases where the number of copies of the targetsequence present in a sample is small, less than approximately 10′copies. Under these conditions it is difficult to distinguish specificattachment of a probe to its target sequence from non-specificattachment of the probe to a sequence different from the targetsequence. One of the solutions to this problem is to use anamplification technique which consists of augmenting the detectionsignal by a preliminary technique designed to specifically andconsiderably increase the number of copies of a target nucleic acidfragment if it is present in the sample.

The articles by Lewis (1992, Genetic Engineering News 12: 1-9) andAbramson and Myers (1993, Curr. Opin. Biotechnol. 4: 41-47) are goodgeneral surveys of amplification techniques. The techniques are basedmainly on either those that require multiple cycles during theamplification process or those that are performed at a singletemperature.

Cycling techniques are exemplified by methods requiring thermo-cyclingand the most widely used of this class of technology is PCR (polymerasechain reaction, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159;European Patent No. 0 201 184) which enables the amplification of aregion of interest from a DNA or RNA. The method usually consists ofthree steps:

(i) dissociating (denaturing) a double-stranded DNA into single-strandedDNAs by heat denaturation/melting (FIG. 1B);

(ii) annealing a primer oligonucleotide to the single-stranded DNA (FIG.1B); and

(iii) synthesizing (extending) a complementary strand from the primer inorder to copy a region of a DNA of interest (FIG. 1C).

After this process is completed the system is heated which separates thecomplementary strands and the process is repeated. Typically 20-40cycles are performed to amplify genomic DNA to the extent that it can befurther analysed.

The majority of exponential nucleic acid amplification processes rely onan excess of upstream and downstream primers that bind to the extreme 3′terminus and the complement of the extreme 5′ end of the target nucleicacid template under investigation as shown in FIGS. 1A-C.

A second class of amplification techniques, known as isothermaltechniques, are those that are performed at a single temperature orwhere the major aspect of the amplification process is performed at asingle temperature. In contrast to the PCR process where the product ofthe reaction is heated to separate the two strands such that a furtherprimer can bind to the template repeating the process, the isothermaltechniques rely on a strand displacing polymerase in order toseparate/displace the two strands of the duplex and re-copy thetemplate. This well-known property has been the subject of numerousscientific articles (see for example Y. Masamute and C. C. Richardson,1971, J. Biol. Chem. 246, 2692-2701; R. L. Lechner et al., 1983, J. BiolChem. 258, 11174-11184; or R. C. Lundquist and B. M. Olivera, 1982, Cell31, 53-60). The key feature that differentiates the isothermaltechniques is the method that is applied in order to initiate thereiterative process.

Broadly isothermal techniques can be subdivided into those methods thatrely on the replacement of a primer to initiate the reiterative templatecopying (exemplified by HDA (Helicase Dependent Amplification),exonuclease dependent amplification (EP1866434), Recombinase PolymeraseAmplification (RPA) and Loop Mediated Amplification (LAMP)) and thosethat rely on continued re-use or de novo synthesis of a single primermolecule (exemplified by SDA (Strand Displacement Amplification andnucleic acid based amplification (NASBA and TMA)).

Recombinase Polymerase Amplification (RPA) is a process in whichrecombinase-mediated targeting of oligonucleotides to DNA targets iscoupled to DNA synthesis by a polymerase (Morrical S W et. Al. J BiolChem. 1991 Jul. 25; 266(21):14031-8 and Armes and Stemple, U.S.application Ser. No. 10/371,641). WO 2008/035205 describes an RPAprocess of amplification of a double stranded target nucleic acidmolecule, comprising the steps of: (a) contacting UvsX, UvsY, and gp32proteins with a first and a second single stranded nucleic acid primerspecific for said double stranded target nucleic acid molecule to form afirst and a second nucleoprotein primer; (b) contacting the firstnucleoprotein primer to said double stranded target nucleic acidmolecule to create a first D loop structure at a first portion of saiddouble stranded target nucleic acid molecule and contacting the secondnucleoprotein primer to said double stranded target nucleic acidmolecule to create a second D loop structure at a second portion of saiddouble stranded target nucleic acid molecule such that the 3′ ends ofsaid first nucleic acid primer and said second nucleic acid primer areoriented toward each other on the same double stranded target nucleicacid molecule without completely denaturing the target nucleic acidmolecule; (c) extending the 3′ end of said first and secondnucleoprotein primers with one or more polymerases capable of stranddisplacement synthesis and dNTPs to generate a first and second doublestranded target nucleic acid molecule and a first and second displacedstrand of nucleic acid; and (d) continuing the reaction throughrepetition of (b) and (c) until a desired degree of amplification isreached.

In order to discriminate amplification of the target from that of futileamplification producing artefacts, probe based systems may be used thatdetect sequences of the amplicon under investigation that are notpresent in the primers supplied to the system.

All of these processes rely only on a template that comprises a bindingsite for the two primers at their extreme termini. A template with thesequalities can be produced by non-specific interactions between theupstream and downstream primers alone and the product (primer-dimers)may be capable of efficient amplification independently of the templateunder investigation, as shown in FIGS. 1D-E. As a consequence of thisfutile amplification, the assay components become consumed bynon-productive events limiting the sensitivity of the assay process.

It is an object of the present invention to provide an alternativeisothermal nucleic acid amplification technique. It is a further objectof the present invention to provide an exponential amplificationtechnique. It is a further object to minimize or eliminate amplificationartefacts and so provide a method for amplifying nucleic acids withincreased specificity and/or sensitivity.

SUMMARY OF THE INVENTION

The present invention thus provides an isothermal process for amplifyinga nucleic acid target molecule that relies on an upstream primer, adownstream primer, a strand invasion system and an oligonucleotide,wherein the upstream and downstream primers are not substrates for thestrand invasion system during the amplification process and do notamplify the target molecule independently of the strand invasion system,wherein the oligonucleotide is a substrate for the strand invasionsystem.

In one embodiment the invention provides an isothermal processcomprising the following steps:

(a) providing an upstream primer, a downstream primer, a strand invasionsystem and an oligonucleotide, wherein the upstream and downstreamprimers are not substrates for the strand invasion system during theamplification process and do not amplify the target moleculeindependently of the strand invasion system, wherein the oligonucleotideis a substrate for the strand invasion system;(b) applying the oligonucleotide to the target molecule and allowing itto invade the duplex thereby rendering some or all of the targetmolecule single-stranded;(c) applying the upstream primer to the single-stranded region of thetarget molecule and extending the 3′ end of the upstream primer withpolymerase and dNTPs to produce a double-stranded nucleic acid targetmolecule;(d) applying the downstream primer to the single-stranded targetmolecule and extending the 3′ end of the downstream primer withpolymerase and dNTPs to produce a further double-stranded nucleic acidtarget molecule;(e) continuing the reaction through repetition of (b) to (d).

In another embodiment the invention provides an isothermal processcomprising the following steps:

(a) providing:

-   -   (i) upstream and downstream primers, each comprising a        single-stranded DNA molecule of less than 30 nucleotides, at        least a portion of which is complementary to sequence of the        target molecule;    -   (ii) an oligonucleotide comprising a single-stranded DNA        molecule of at least 30 nucleotides, at least a portion of which        is complementary to sequence of the target molecule intervening        the forward and reverse primers, and optionally further        comprising a downstream element at its 3′ terminus which is        complementary to sequence of the target molecule and which is        not a polymerase substrate;        (b) contacting the oligonucleotide (ii) with recombinase to        enable it to invade the complementary region of the target        molecule thereby rendering the complementary region of the        target molecule and adjacent regions single-stranded;        (c) applying the upstream primer to the single-stranded region        of the target molecule and extending the 3′ end of the upstream        primer with polymerase and dNTPs to produce a double-stranded        nucleic acid target molecule;        (d) applying the downstream primer to the single-stranded target        molecule and extending the 3′ end of the downstream primer with        polymerase and dNTPs to produce a further double-stranded        nucleic acid target molecule;        (e) continuing the reaction through repetition of (b) to (d).

Advantageously these methods provide isothermal and exponentialamplification of a target nucleic acid molecule. The amplificationmethods are more specific and sensitive than known methods and result inminimal or no amplification artefacts.

Preferably the strand invasion system comprises a recombinase system,such as the T4 UvsX/gp32/UvsY system. Preferably at least a portion ofthe oligonucleotide is complementary to a portion of the target sequenceintervening the upstream and downstream primers. Preferably theoligonucleotide comprises a single-stranded DNA molecule of at least 30nucleotides which may have a non-extendable 3′ terminus.

Preferably the substrate for the strand invasion system facilitates theseparation of the target template duplex or the product of primerextension onto the target nucleic acid. One or more additionaloligonucleotides may facilitate the separation of the target duplex bythe intervening oligonucleotide. Preferably the oligonucleotidecomprises a downstream element at its 3′ terminus which is complementaryto the target sequence and which is not an efficient polymerasesubstrate. This may bind to the target strand released by theoligonucleotide and branch migrate into the proximal duplex nucleic acidfurther separating the duplex strands.

In another embodiment the invention provides a kit for isothermallyamplifying a nucleic acid target molecule comprising an upstream primer,a downstream primer, a strand invasion system and an oligonucleotide,wherein the upstream and downstream primers are not substrates for thestrand invasion system during the amplification process and do notamplify the target molecule independently of the strand invasion system,wherein the oligonucleotide is a substrate for the strand invasionsystem

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F show the formation of primer dimers in a primer dependentamplification reaction.

A: Upstream and downstream primers are incubated with a duplex template.The template is cognate at its extreme ends to the primers. One stand ofthe template is cognate to the upstream primer and the other strand iscognate to the downstream primer.

B: The template strands are separated which allows the upstream anddownstream primer to bind.

C: Extension of the template bound primers produce two identicalduplexes. Each duplex may participate in the previous steps in thereaction such that the template is exponentially amplified.

D-E: The upstream and downstream primer copy onto each other in theabsence of template. These may replace the template under investigationand can also be exponentially amplified causing an artefact.

FIGS. 2A-2E demonstrate the basic amplification system of this inventiontogether with an optional probe based detection system.

FIGS. 3A-3D show an amplification method where a downstream element isused to protect from non-specific amplification products.

FIGS. 4A-4D show an amplification method utilizing a reverse complementoligonucleotide such that non specific products cannot be formed.

FIGS. 5A-5E show the sequence of events that can lead to primerartefacts in a tripartite system.

FIGS. 6A and 6B shows template/primer configurations. A: template/primerconfigurations;

B shows amplification in a 2-primer system (measured by Sybr Greenfluorescence).

FIGS. 7A and 7B show template/primer/oligonucleotide configurations. A:a schematic of template/primer/oligo configurations;

B shows amplification in a tripartite system (measured by Sybr Greenfluorescence).

FIG. 8 shows the effect of primer length in a tripartite system.

FIGS. 9A and 9B show template/primer/oligonucleotide configurationsincluding downstream 2-O-methylated oligonucleotides and probes. A: aschematic of template/primer/oligo configurations;

B: shows the effect of using an intermediate oligonucleotide having amethylated downstream element.

FIG. 10 shows that a tripartite system can amplify from biologicallyderived DNA.

FIGS. 11A and 11B show the result of amplification with a tripartitesystem using 75 nM of downstream methylated intermediateoligonucelotide. A: amplification versus time;

B shows that the sensitivity of the tripartite system using a downstreammethylate intermediate can be at the level of a single molecule.

FIG. 12 shows the use of crowding agents in tripartite system.

FIG. 13 shows amplification in a tripartite system interrogated byprobes.

DETAILED DESCRIPTION OF THE INVENTION

The invention describes a method that enables isothermal and exponentialamplification of a target nucleic acid. The nucleic acid sequence maycomprise DNA, reverse transcribed RNA (cDNA) or genomic DNA. The nucleicacid may also contain modified or unnatural nucleosides where they canbe copied by the action of a polymerase.

In contrast to other nucleic acid amplification processes, the upstreamand downstream primers that bind to the extreme termini of the nucleicacid (terminal primers) are, alone, not able to induce exponentialamplification of the target nucleic acid. The exponential aspect of theamplification is enabled by one or more oligonucleotides (intermediateor intervening oligonucleotides, IO) that are cognate to a proportion ofthe template sequence intervening the upstream and downstream primers.Since a template cognate to the upstream and downstream primers alone isnot a viable amplification unit, the system can be designed such that itis impervious to loss of sensitivity by the primer dimer artefactsdescribed in FIGS. 1A-1F and also other mispriming artefacts.

The primers are not able to amplify in the absence of the IO sequenceand preferably the IO is non-extendable. As a result artefactualamplification is abolished or significantly reduced since the IO cannotimpart artefactual amplification in its own right. Furthermore in someaspects of the invention, the IO comprises sequences that are notsubstrates for a polymerase and as such there are no artefactual eventsthat can reproduce the amplifiable sequence in the absence of the targetunder investigation.

In one aspect the invention provides an isothermal process foramplifying a nucleic acid target molecule that relies on an upstreamprimer, a downstream primer, a strand invasion system and anoligonucleotide, wherein the upstream and downstream primers are notsubstrates for the strand invasion system during the amplificationprocess and do not amplify the target molecule independently of thestrand invasion system, wherein the oligonucleotide is a substrate forthe strand invasion system.

In a further embodiment the invention provides an isothermal process foramplifying a double-stranded nucleic acid target molecule comprising thefollowing steps:

(a) providing an upstream primer, a downstream primer, a strand invasionsystem and an oligonucleotide, wherein the upstream and downstreamprimers are not substrates for the strand invasion system during theamplification process and do not amplify the target moleculeindependently of the strand invasion system, wherein the oligonucleotideis a substrate for the strand invasion system;(b) applying the oligonucleotide to the target molecule and allowing itto invade the duplex thereby rendering some or all of the targetmolecule single-stranded;(c) applying the upstream primer to the single-stranded region of thetarget molecule and extending the 3′ end of the upstream primer withpolymerase and dNTPs to produce a double-stranded nucleic acid targetmolecule;(d) applying the downstream primer to the single-stranded targetmolecule and extending the 3′ end of the downstream primer withpolymerase and dNTPs to produce a further double-stranded nucleic acidtarget molecule;(e) continuing the reaction through repetition of (b) to (d).

A preferred isothermal process for amplifying a double-stranded nucleicacid target molecule comprises the following steps:

(a) providing:

-   -   (i) upstream and downstream primers, each comprising a        single-stranded DNA molecule of less than 30 nucleotides, at        least a portion of which is complementary to sequence of the        target molecule;    -   (ii) an oligonucleotide comprising a non-extendable,        single-stranded DNA molecule of at least 30 nucleotides, at        least a portion of which is complementary to sequence of the        target molecule intervening the forward and reverse primers;        (b) contacting the oligonucleotide (ii) with recombinase to        enable it to invade the complementary region of the target        molecule thereby rendering the complementary region of the        target molecule and adjacent regions single-stranded;        (c) applying the upstream primer to the single-stranded region        of the target molecule and extending the 3′ end of the upstream        primer with polymerase and dNTPs to produce a double-stranded        nucleic acid target molecule;        (d) applying the downstream primer to the single-stranded target        molecule and extending the 3′ end of the downstream primer with        polymerase and dNTPs to produce a further double-stranded        nucleic acid target molecule;        (e) continuing the reaction through repetition of (b) to (d).

The inventive methods rely on the following components.

Upstream primer (or forward primer) binds to one strand of the targetnucleic acid molecule at or proximal to the 5′ region of the interveningoligonucleotide (TO).

Downstream primer (or reverse primer) binds to one strand of the targetnucleic acid molecule at or proximal to the 3′ terminus of the IO. Itbinds to the opposite strand to which the upstream primer binds.

Essentially, a primer binds to a template and is extended by the actionof a polymerase. The forward and reverse primers must be efficientpolymerase substrates. In some aspects, when used in conjunction with arecombinase system, the primers should not be competent recombinasesubstrates. This means that they should be less than 30 nucleotides inlength. Preferably the primer is less than 25 nucleotides. Mostpreferably the primer is approximately 15 to 23 nucleotides. The primersare capable of binding to opposite strands of the target nucleic acidmolecule. It is not essential that the entire primer binds to (iscomplementary with) the target sequence.

Intervening or intermediate oligonucleotide (IO) is a substrate for thestrand invasion system (SIS). The substrate for the strand invasionsystem facilitates the separation of the target template duplex or theproduct of primer extension onto the target nucleic acid, therebyallowing the primers access to bind to complementary single stranded DNAon the target molecule.

In one embodiment the IO may comprise Peptide Nucleic Acid (PNA) whichis able to invade double stranded DNA molecules without recourse to arecombinase. In this embodiment the PNA fulfils the role of both theoligonucleotide and the strand invading system.

In a preferred embodiment the strand invasion system comprises arecombinase system. In this embodiment at least a portion of theoligonucleotide should be complementary to a portion of the targetsequence intervening the upstream and downstream primers. Theintervening oligonucleotide should comprise a region that is arecombinase substrate. As recombinases preferentially effect a substrateoligonucleotide that is more than about 30 nucleotides (Formosa T. andAlberts B., JBC, (261) 6107-6118, 1986; Gamper B et al., Biochemistry,(42) 2643-2655, 2003), preferably the oligonucleotide comprises asingle-stranded DNA molecule of at least about 30 nucleotides or a DNAsequence and a modified sequence that are together more than 30nucleotide bases. Further, it should preferably have a cognate area longenough to invade a template efficiently and so at least a portion of theIO should be complementary to the target sequence intervening theforward and reverse primers. Generally this is a minimum of 24 bases andoptimally around 38 bases. The 5′ portion of the complementary sequenceis preferably close enough to the duplex terminus that the meltingtemperature of the residual duplex results in dissociation of theresidual duplex after binding. Usually this means that the 5′ terminusof the complementary sequence should be no more than 15-20 nucleotidesfrom the duplex terminus.

The IO may also comprise a 5′ terminus that is not cognate to thetemplate in order to efficiently seed the cognate area with recombinase.Typically this would be in excess of 12 bases. Thus the total length ofthe IO is preferably at least 36 bases, more preferably at least 50bases, including the cognate region. It may also comprise a 3′ terminusthat is not cognate to the template.

It is preferred that the 10 has a non-extendable 3′ terminus. This maybe achieved by incorporating one or more of several modifiednucleotides. Typically these will incorporate a 3′ modification of theterminal nucleotide. Examples of these modified nucleotides aredideoxynucleotide nucleotides, 3′ amino-allyl, 3′-carbon spacers ofvarious lengths, nucleotides incorporated in a reversed orientation(3′-3′ linkage), 3′ phosphate, 3′biotin, 3′ salyl, 3′-thiol.Alternatively the terminus may comprise nucleotides incompatible withextension by a polymerase due to their poor substrate capability such asPNA or LNA or 2′-5′-linked DNA or 2′-O-methyl RNA.

Recombinase Systems

As mentioned above, preferably the strand invasion system comprises arecombinase system. Recombinases should bind to DNA molecules longerthan about 30 nucleotides. Preferably, they have a strong preference forsingle-stranded DNA and a relatively weaker preference fordouble-stranded DNA. In the inventive method this allows them to bind tothe IO but not to the upstream or downstream primers.

Various recombinase systems are known to those familiar with the art andhave been variously reviewed (e.g. Piero R. Bianco et al. Frontiers inBioscience 3, d570-603, 1998, 570 DNA Strand Exchange Proteins: ABiochemical and Physical Comparison). Any recombinase system may be usedin the method of the invention and the detailed application ofrecombinases for the invasion of nucleic acid duplexes is known to thosefamiliar with the art (Kodadek T et. Al., JBC 264, 1989; and Liu J, JBCNa Qianl, 281, 26308-26319, 2006).

The recombinase system may comprise a components derived from yeast,bacteria phage or mammals or other eukaryotes. The recombinase systemmay be mesophilic or thermophilic. For example, the recombinase may bederived from a myoviridae phage. The myoviridae phage may be, forexample, T4, T2, T6, Rb69, Aeh1, KVP40, Acinetobacter phage 133,Aeromonas phage 65, cyanophage P-SSM2, cyanophage PSSM4, cyanophageS-PM2, Rb14, Rb32, Aeromonas phage 25, Vibrio phage nt-1, phi-1, Rb16,Rb43, Phage 31, phage 44RR2.8t, Rb49, phage Rb3, or phage LZ2. In apreferred embodiment, the T4 recombinase UvsX may be used. The Radsystems of eukaryotes or the recA-Reco system of E. Coli or otherprokaryotic systems may also be used.

Usually a recombinase will polymerise onto a single-strandedoligonucleotide in the 5′-3′ direction. The invention as describedherein relates to such a recombinase. However, the recombinase maypolymerise in a 3′-5′ direction and such recombinases may also be usedin the method of the invention. In this case and with reference to thedirectionality of the components described, the reverse applies.

Recombinase accessory proteins may be included in the system, such assingle-stranded binding protein (e.g. gp32) and recombinase loadingagent (e.g. UvsY). In a preferred embodiment, the recombinase systemcomprises the T4 gp32, UvsX and UvsY. When such a system is used, allsingle stranded elements (i.e. primers and IO) become coated with thesingle stranded binding protein (e.g. gp32). The recombinase loadingagent (e.g. UvsY) acts as a cofactor for the recombinase and coats theTO. The recombinase (e.g. UvsX) competently coats only the IO since onlythis element comprises a sufficient length to induce the process.

The recombinase (e.g. UvsX), and where used the recombinase loadingagent (e.g. UvsY) and single strand DNA binding protein (e.g. gp32), caneach be native, hybrid or mutant proteins from the same or differentmyoviridae phage sources. A native protein may be a wild type or naturalvariant of a protein. A mutant protein (also called a geneticallyengineered protein) is a native protein with natural or manmademutations such as insertions, deletions, substitutions, or a combinationthereof, that are at the N terminus, C terminus, or interior (betweenthe N terminus and the C terminus). A hybrid protein (also called achimeric protein) comprises sequences from at least two differentorganisms. For example, a hybrid UvsX protein may contain an amino acidfrom one species (e.g., T4) but a DNA binding loop from another species(e.g., T6). The hybrid protein may contain improved characteristicscompared to a native protein. The improved characteristics may beincreased or more rapid amplification rate or a decreased or morecontrollable amplification rate.

Other factors used to enhance the efficiency of the recombinase systemmay include compounds used to control DNA interactions, for exampleproline, DMSO or crowding agents which are known to enhance loading ofrecombinases onto DNA (Lavery P et. Al JBC 1992, 26713, 9307-9314;WO2008/035205). Whereas crowding agents such as PVA, gelatine or albuminare known to influence enzyme kinetics by increasing the effectiveconcentration of reagents due to volume occupation (Reddy M K et. Al.Methods Enzymol. 1995; 262:466-76; Harrison B, Zimmerman S B. AnalBiochem. 1986 Nov. 1; 158(2):307-15; Reddy M K, Weitzel S E, von HippelP H. Proc Natl Acad Sci USA. 1993 Apr. 15; 90(8):3211-5; Stommel J R et.Al. Biotechniques. 1997 June; 22(6):1064-6), DMSO, betaine, proline anddetergents may enhance the systems by altering the Tm or secondarystructure of the oligonucleotides in the assay.

Polymerase

The polymerases used in the process of the invention are preferablythose with strand displacement activity. This activity is a well-knownproperty of certain DNA polymerases (Sambrook et al., 1989, MolecularCloning: A Laboratory Manual, 2nd edition, pp. 5.33-5.35, Cold SpringHarbor Laboratory, Cold Spring Harbor). The properties of the DNApolymerases, particularly the strand displacement activity of some ofthem, are given in detail by Kornberg and Baker, DNA Replication, 2ndedition, pp. 113-225, Freeman, N.Y. (1992). Strand displacement is not aproperty common to all DNA polymerases since some of them, like T4 DNApolymerases, are not capable of accomplishing strand displacement alone.Strand displacement may in these cases be imparted by the addition ofthe polymerases accessory proteins. Strand displacement activity wasshown initially for the Klenow fragment of Escherichia coli DNApolymerase I (Masamune and Richardson, 1971, J. Biol. Chem. 246:2692-2701), which confers on this enzyme the capability of initiatingreplication of nucleic acid from the 3′OH end of a cleavage site in adouble-stranded DNA. This strand displacement activity has also beenshown in thermostable DNA polymerases such as Tli DNA polymerase (Konget al., 1993. J. Biol. Chem. 268: 1965-1975). In this case it has alsobeen shown that mutated forms of this enzyme do not have exonuclease5′-3′ activity that has a higher strand displacement capacity. Thisstrand displacement activity has also been shown for T7 DNA polymerase(Lechner et al., 1983. J. Biol. Chem. 258: 11174-11184) and for HIVreverse transcriptase (Huber et al., 1989, J. Biol. Chem. 264:4669-4678).

Preferably, a DNA polymerase with no 5′-3′ exonuclease activity is usedto accomplish the amplification cycle according to the invention sincethe effectiveness of the strand displacement activity is greater inenzymes with no such exonuclease activity. The Klenow fragment ofEscherichia coli DNA polymerase I is an example of a polymerase with no5′-3′ exonuclease activity, as are polymerases such as T7 DNA polymeraseor Sequenase (US Biochemical). T5 DNA polymerase or Phi29 DNA polymerasecan also be used. However, a DNA polymerase having this 5′-3′exonuclease activity can be used when it does not prevent theamplification process from being carried out. In this case, the yield ofthe amplification reaction can be improved by specific inhibition of the5′-3′ exonuclease activity of DNA polymerases under the reactionconditions employed.

Stand displacement may also be enhanced by the application of enzymesystems or other elements that stabilise single stranded rather thanduplex DNA. Examples of such systems are the application of DNAhelicases, stabilisation by single stranded binding proteins as well asthe influence of the particular polymerase used in the system. It isessential that the method of enhancement of strand displacement does notinterfere with the strand invasion system.

Suitable polymerases include polI or polI fragments or variants such asthose from E. Coli, Bacilus bubtilis, stearothermophilus or T7polymerases. Equally a polymerase holoenzyme complex can be used such asthat described for phage T4. Preferred polymerases are klenow, exo-, aT7 polymerase or BST phi29 or pol-I of Bacillus subtilis or aholoenzyme. In some embodiments and especially where a downstreamelement or a reverse complement are used it may be preferable that thepolymerase does not have strong strand displacing activity as is thecase for the klenow fragment of E. Coli polI. In other embodimentsstrong strand displacement activity of the polymerase may be anadvantage.

ATP Regeneration System

Where a recombinase is utilised for the strand invasion step the systemmay have a requirement for a source of energy. The majority of theseenzymes utilize ATP as the energy source but since ATP collatesmagnesium ions necessary for enzyme activity it is prudent to supply anadditional ATP regeneration system rather than raise the concentrationof ATP. ATP generation systems may involve various enzymes in theglycolytic or other biochemical pathways and together with ATPconsumption these enzymes together with the SIS enzymes induceorthophosphate and or pyrophosphate accumulation concomitant with theproduction of AMP and ADP. The accumulation of inorganic phosphates isalso able to chelate magnesium and may be deleterious to the system innumerous ways. The conversion of pyrophosphate to orthophosphate may beachieved by pyrophosphatases and orthophosphate conversion to lessharmful organophosphates has also been variously reported.

Preferably the inorganic phosphate or orthophosphate conversion toorganophosphate utilises sucrose and sucrose phosphorylase or othersugar phosphorylases. Alternatively nicotinamide riboside and purinephosphorylase may be used.

Since some ATP regeneration systems utilise ADP exclusively as asubstrate, it may be an advantage to convert AMP produced by somerecombinases to ADP using a myokinase. This also avoids prematuredepletion of the ATP resource. Under standard operating conditionsdescribed in the examples, the T4 recombinase does not produce AMP andthis step can be omitted.

The ATP regeneration systems themselves generally use phosphocreatinewith creatine kinase or phospho-phenyl-pyruvate and pyruvate kinase.Since the UVSX recombinase may burn up to 300 molecules of ATP in oneminute and since 3 μM UVSX may be used, it is advantageous to use asystem with 40-100 mM phosphocreatine.

In practice it may be advantageous to include one or more of the aboveenergy sources in the reaction solution, for example, one or more ofATP, Phosphocreatine, Creatine Kinase, Myokinase, Pyrophosphatase,Sucrose, Sucrose phosphorylase.

General Considerations for Optimisation of the System

In practice when carrying out the method of the invention standardtitration of the various components of the system shown in the standardoperating procedure may be required to ensure optimal amplification.Titration of components includes proteinaceous metal ion and salttitrations. In the case of salts the nature of the cation and anion aswell as the optimal concentration may be assessed to achieve optimalamplification.

Thus, various components may be included such as: magnesium ions;phosphocreatine and its counterion, pH adjusters, DTT or other reducingagents, BSA/PEG of various molecular weight distributions or othercrowding agents, ATP and its counter ion, dNTP, Sucrose, CreatineKinase, Myokinase, Pyrophosphatase, Sucrose Phosphorylase, UvsX, UvsY,gp32 (NEB, 10 mg/ml), Klenow, exo- or other polymerases.

The buffer system utilised in the amplification protocol should becompatible with all elements supplied to the system. Clearly optimumconditions for all components may not be achieved within a singlebuffered system. Numerous opportunities exist that may be used tobalance the experimental condition such that the system worksefficiently.

Primer and oligonucleotide design also impacts on the balance of thesystem since alteration in the length and melting temperature of thevarious primers and oligonucleotides, as well as the chosen ampliconlength, can alter the balance of duplex separation and primer extension.

Melting temperature (Tm) is temperature that half of the population ofidentical duplex is separated. The length and sequence of a duplexrelates to the Tm such that a longer duplex tends to have a higher Tm.Equally the buffer solution utilised during amplification may alter Tmsince various salts and other components may alter the affinity betweentemplates and primers (Chadalavada S. V. FEBS Letters 410 (1997)201-205). In the context of this invention the Tm relates to the areasof a duplex that have not been invaded by an IO. Although the detailherein relates to systems developed to work at approximately 40° C., itis possible to develop systems that function at differing temperatures,for example from about 21 to 50° C., preferably from about 25 to 45° C.,most preferably from about 37-40° C. Consequently the lengths andsequences of the target and primers may be adjusted accordingly. Wherethe template under investigation is negatively supercoiled then thesetendencies do not apply to the initial template since negativelysupercoiled DNA can be treated as if it is single stranded DNA. In thefirst round of amplification it may be necessary to heat or otherwisechemically/enzymatically denature or cleave the target to initiate theamplification process. An additional primer called a bumper primer maybeused to initiate the first round of amplification as previously reported(Nuovo G. J; Diagn Mol Pathol. 2000 (4):195-202). Furthermore if thesystem enables the upstream or the downstream primer to extend slowly inthe first rounds of amplification then no additional features arenecessary but there will be a lag phase caused by the resistance to theinitial amplification event.

Amplification Method

The following part of the description will describe one embodiment ofthe invention wherein the method relies on a strand invasion system(SIS) induced by a recombinase. A recombinase reacts with a singlestranded oligonucleotide substrate and enables it to invade acomplementary strand within a duplex nucleic acid, displacing the otheroutgoing strand (OS) of the duplex.

The essential principal of the invention is that an oligonucleotide (TO)is presented that invades a duplex nucleic acid target. The consequenceof this event is that the strands of the target duplex are separated anddissociate in the template region cognate to the invadingoligonucleotide but also in an area outside but proximal to the invasionsite and this allows terminal primers to bind to the component strands,and extend, which result in two duplex copies. The process repeatsitself recursively with resultant exponential amplification of thetarget.

In one embodiment of the invention a duplex target nucleic acid isinvaded in its mid region by a single stranded non-extendibleoligonucleotide (TO) by the action of a recombinase. Invasion by theoligonucleotide disturbs the duplex stability to the extent that theduplex falls apart and becomes single stranded. This exposes bindingsites for upstream and downstream primers (that are not recombinasesubstrates) and these extend onto the separated strands creating twocopies of the duplex (FIG. 2).

FIGS. 2A-2E demonstrate the basic amplification system of the inventiontogether with an optional probe-based detection system. This system isprotected from non-specific amplification to some extent butnon-specific products may be formed after prolonged incubation as shownin FIGS. 5A-5E (discussed below). FIGS. 2A to 4D are described inconnection with the T4 gp32, UvsX and UvsY recombinase system but anyrecombinase system, or other strand invasion system may be used.

2A: The elements of this system are shown as I-IV. A duplex template isshown as dotted lines. I represents an upstream primer that is not asubstrate or is only a minimal substrate for the SIS as described (i.e.it does not bind recombinase). II shows the central component of thesystem, the TO, which can be extendable by the action of a polymerase ornon-extendable. This nucleic acid element is a substrate for the SIS inthat it binds recombinase and invades the target duplex. It does notneed to be acted on by a polymerase. This element may also optionallycomprise an extended 3′ or 5′ tail that is not cognate to the sequencethat is invaded. The 5′ region of the cognate area of the invadingoligonucleotide is close enough to the duplex terminus such that themelting temperature of the residual duplex is below the ambienttemperature of the system and results in dissociation of the residualupstream duplex after binding. III shows the potential site for anoptional probe system such as a T7 exonuclease sensitive probe or amolecular beacon. IV represents the downstream primer that is not asubstrate or only a minimal substrate for the SIS.

All single stranded elements of the system become coated with the singlestranded binding protein GP32. UVSY which is a cofactor for UVSX alsocoats the IO. The GP32 coated elements have a reduced capacity forbranch migration. UVSX competently coats only the IO since only thiselement comprises a sufficient length to induce the process.

FIG. 2B After coating the IO (II) with recombinase, it is able to invadethe duplex. The duplex separates and becomes single-stranded in theregion of invasion and also in adjacent regions, usually around 15 to 20nucleotides in length. This releases the upstream terminal end of theduplex nucleic acid

FIG. 2C: The upstream primer (I) is able to bind to the released strand.Primer concentration temperature and other system components such asdenaturants are optimised such that the primer binds efficiently despiteits proximity to its melt temperature.

FIG. 2D: The upstream primer (I) extends which stabilizes its productand displaces the IO (II). This recreates the original duplex. Thedownstream primer (IV) together with the optional probe (III) is able tobind to the displaced downstream region. The system can be optimisedsuch that the upstream or downstream primer concentrations areasymmetric and by this mechanism an excess of the downstream primerensures that a single stranded excess of downstream product is inducedat the end of the reaction and that the binding site for the probe isavailable

FIG. 2E: The downstream primer extends doubling the duplex number.

Usually two complementary oligonucleotides bind together with anaffinity that depends on the length and sequence of the cognate region.The two strands tend to fall apart only above a particular temperatureand this is called the melt temperature (Tm). The length of the cognateregion is proportional to the Tm. The Tm is also affected by themagnesium and monovalent salt concentration and is also reduced in thepresence of single stranded binding proteins. The transitory presence ofa recombinase on an oligonucleotide will increase its Tm. The relevantmelt temperature parameters are therefore generally assessedempirically.

A duplex may be invaded by a recombinase-coated oligonucleotide and thisis dependent on it being cognate to one strand of the invaded duplex.The other strand of the duplex is nominated as the outgoing strand (OS)and is essentially separated from the cognate strand of the duplex andbecomes single-stranded. Consequently where the residual duplex outsidethe invaded region has a length and sequence such that the Tm is belowthe ambient temperature then the complete duplex will dissociateproducing two single stranded termini which can bind terminal primers.

Most recombinases polymerise onto an oligonucleotide from its 5′ regiontowards its 3′ region and once the coated oligonucleotide invades aduplex then the SIS continues to polymerise onto the duplex 3′ to theinvading oligonucleotide (downstream). The coated elements of an invadedsystem are held together more tightly than an uncoated region. It isalso notable that a primer bound to a template that is coated with arecombinase cannot be extended until the recombinase has depolymerisedand has been removed.

As a consequence of the above observations, if the template duplexterminus upstream of the IO is close to the invaded region, then theterminus will separate since it is not coated with the recombinase and aprimer may bind and extend displacing the IO such that it can be re-usedby the system. Under the same circumstances, the downstream terminuswill be held together until the recombinase depolymerises/falls offwhich is also in a 5′ to 3′ direction. If the Tm of the downstreamterminus is higher than the ambient temperature then its strands willremain associated even after depolymerisation of the recombinase but thestrands will still be separated as the upstream primer extends anddisplaces the strands of the original duplex. This will enable adownstream primer to subsequently bind and extend (FIGS. 2A-2E).

If the upstream terminus is not close to the invaded region but theproximity of the downstream terminus is close enough to allow meltingthen it might be expected that the downstream terminus would separateafter depolymerisation of the recombinase. Surprisingly this is not thecase since the closed upstream terminus of the duplex branch migrates asthe recombinase depolymerises displacing the invading oligonucleotide,repositioning the outgoing strand onto its partner and reforming theoriginal duplex and does not give an opportunity for the downstreamprimer to bind. Branch migration is rapid in this scenario since theoutgoing strand remains wrapped around the complex in a plectonemicconformation and remains highly associated with its cognate strand evenwhen displaced.

The consequence of these events is that for a system to be viable suchthat the SIS enables a duplex to be separated then the upstream terminusof a target duplex must be separated during the strand invasion event.This is achieved by ensuring that the upstream region of the duplexproximal to the invasion site has a melting temperature close to orbelow the ambient temperature. This is easily determined by the skilledperson using standard techniques but will be influenced by systemcomponents such as single stranded binding proteins, metal ionconcentrations and salt concentration.

Accordingly, the IO should preferably be designed such that it iscomplementary to the target molecule leaving only about 10-20 bases,preferably about 15-17 bases, on either side of the cognate region.Thus, for example, where the application is performed at 40° C. a nonextendible IO is supplied to the system such that it leaves 10-17 basesof duplex on either side of the cognate region. On invasion by the IOthe upstream terminus (in relation to the invading oligonucleotide) ofthe duplex melts whereas the 3′ terminus is held together due to theinvasion dependent polymerisation of the recombinase onto the downstreamduplex. The upstream primer binds to the melted upstream terminus of theduplex and is extended, thereby displacing the TO. The downstream end ofthe duplex can either be separated by the continued extension of theupstream primer or it may be short enough so that when the recombinasedepolymerises it also melts.

The scenario above depicts the consequence of a recombinase thatpolymerises in the 5′-3′ direction (upstream to downstream). Thedescription herein describes amplification with this type ofrecombinase. Where a recombinase is utilised that polymerises in theopposite direction then the configuration of the system is reversed.Most recombinases prefer a region of 12-15 bases at the upstreamterminus of an IO to facilitate seeding of the recombinase and thisfeature may be incorporated such that an IO comprises an upstreamnon-cognate region.

In the above-described methods, although amplification artefacts areminimized, it is plausible that a primer could non-specifically copyonto the IO and that the product of this extension could be displaced,copying onto a further primer as shown in FIGS. 5A-E.

FIGS. 5A-5E shows the mechanism by which primer artefacts can occur in atripartite system that does not include a downstream element.

FIG. 5A: The system components that induce artefactual amplificationcomprise the upstream primer (I), the intermediate oligonucleotide (II)and the downstream primer (III).

FIG. 5B: A downstream primer may occasionally copy onto the intermediateoligo.

FIG. 5C: Any additional oligonucleotide primer may copy onto theintermediate upstream of the position that the downstream primeroccupied.

FIG. 5D: Extension of the additional oligonucleotide primer will resultin the displacement of the product of the downstream primer extension.

FIG. 5E: Finally, if an upstream primer copies onto the displacedproduct then an amplifiable unit may be produced.

These events are more complex than those involved in the production ofprimer dimer artefacts for the two primer system described in FIG. 1 andas a result the sensitivity of the system for assessing the presence oftest template is improved over systems dependent on only two primers.

Although such an event is rare the consequent oligonucleotide sequencewould potentially be an amplifiable unit. However, this eventuality isabrogated by embodiments of the invention discussed below. In order toovercome any potential for non-specific amplification the followingphenomenon may be utilised.

Where only one end of the duplex is separated due to the invasion by theIO then a cognate primer can bind to the dissociated terminus but thepartially tethered outgoing strand remains in close proximity. Since theoutgoing strand also comprises an identical region to that of the primerit is able to compete with the incoming primer for binding to thetemplate. Additionally the tethered terminus may also branch migrateafter depolymerisation of the recombinase, reinstating the originalduplex before a primer bound to the separated terminus has anopportunity to extend. Under these circumstances and where only theupstream terminus is melted then extension of the upstream primer may becompromised and become dependent on the separation of the downstreamtermini such that the strands of the duplex are no longer tethered andfall away. By this process, competition by the outgoing strand isabolished. A system can therefore be designed such that despite theseparation of the upstream aspect of the duplex, the upstream primer isonly competent if the downstream termini have also been separated. Thedependence of the system on the separation of both termini addsspecificity to the system and can be achieved by altering the competencyof the upstream primer in favour of reinstatement of the outgoing strandor by using a polymerase at low concentrations or with weak stranddisplacement activity (Klenow exo-activity) as this will also effect thebalance of primer extension with duplex reinstatement. Under anycircumstances it is found that amplification is substantially fasterwhere the two strands of the duplex are separated. Therefore artefactualamplification, that does not impart this quality, will be outpaced ifthe amplification of the specific target induces strand separation.

The competence of the upstream primer can be altered by severalmechanisms, for example:

(i) The upstream primer may be designed to overlap with the invading IO.The region of overlap is preferably about 5 to 10 nucleotides. Extensionof an overlapping primer will rely on the preliminary branch migrationof the primer onto the template displacing the IO. Under somecircumstances, and specifically where single stranded binding proteinsare present, branch migration is slow and pauses the extension of theprimer such that competition between the binding and extension of primerversus the re-instatement of the original duplex may be in favour ofreinstatement. Another advantage of this embodiment is that the terminiproximal to the IO do not separate if the length is above 18-23 bases.As such the melting temperature is above the ambient temperature of thereaction where this is about 40° C. The primer described in thisconstruct has a Tm that is above this figure but creates a terminus witha Tm below the ambient temperature. If such a primer is used then it canbind to the cognate terminus of the melted duplex and its 3′ region willbranch migrate to onto the invaded aspect of the duplex if it is cognateand subsequently extend by the action of a polymerase. If the primerbecomes part of a non-specific/template independent product then unlessit is positioned perfectly with the IO region, it may not be a viableamplification unit.

(ii) The upstream primer may be temporarily blocked and rely on anenzymatic cleavage prior to extension. This is exemplified by a 3′blocked primer comprising an RNA base proximal to the 3′ terminustogether with RNAse H. Analogous systems using alternative endonucleasesmay be used as can any mechanism that slows the progress of the primer.

In the embodiment described above the amplification becomes dependent onthe melting of the downstream terminus as well as the upstream terminus.Systems designed to rely on melting of the downstream terminus can addabsolute specificity to the amplification. If the Tm of a downstreamprimer is higher than the ambient temperature then the downstreamterminus will not melt and furthermore non-specific artefactual productwill not amplify. It is an advantage to use such primers but the problemremains as to how a primer with a Tm above the ambient temperature ofthe reaction can induce a terminus that will separate. This isaccomplished by utilizing other additional sequence dependent elementsto melt the downstream terminus.

For example, the downstream tethered termini may be separated by thebinding of one or more additional oligonucleotides which facilitate theseparation of the target duplex by the intervening oligonucleotide andwhose function depends on the IO invasion step. The value of thisapproach is that such an oligonucleotide is designed to bind andseparate the duplex but is neither a polymerase nor a recombinasesubstrate. Such an oligonucleotide cannot participate in the productionof a primer artefact since it is not a polymerase substrate which isimportant as shown in FIG. 5. Preferably the additional oligonucleotidebinds to the strand released by the IO and branch migrates into theproximal duplex nucleic acid. Importantly where its function demandsbranch migration and where branch migration is found to be inhibited bysingle stranded binding proteins then it can be designed such that itdoes not bind significantly to single stranded binding proteins. Thisapproach is exemplified by:

(i) The IO may comprise sequence which is an extension of its 3′terminus (downstream element, DE) which is cognate to the targetterminal region, as shown in FIGS. 3A-3D and discussed below.

In this embodiment, the DE comprises elements that are not a polymerasesubstrate and optionally neither recombinase nor SSB substrates.Typically this may be imparted by the use of 2′ modified nucleotides.Typical modifications of the 2′ position include hydroxylation,methylation and alkylation. It may alternatively be induced bymodification of the base sugar or phosphate component that results intemplate and/or primer incompetent qualities. Suitable elements includeRNA and RNA analogues, such as locked nucleic acid (LNA), morpholino,peptide nucleic acid (PNA) and other nucleic acid modifications thatenable hybridization. These oligonucleotides differ as they have adifferent backbone sugar but still bind according to Watson and Crickpairing with RNA or DNA, but cannot be amplified as polymerase is unableto recognise them. It is important that the element is able to hybridiseto its target sequence.

The DE may be an extension of the IO and is able to branch migratedownstream of the invading section disturbing the remaining duplex andleaving an intact area of duplex with a Tm below the ambienttemperature, separating the duplex such that a primer can bind andextend. There is a cognate overlap between the downstream primer and theDE. It is important that the DE and downstream primer cannot copy ontoeach other and as such the DE should be neither a primer, nor a templatesubstrate for a polymerase, i.e. it should not allow a primer to bindand extend upon this region beyond its junction with the IO and onto theIO. The 3′ terminus of the DE may optionally have additional spuriousbases and/or be blocked from extension to facilitate this, for exampleby placing a non-extendible unit at it 3′ terminus or by the addition ofnon-cognate bases in this region. Typically extension of the 3′ terminusis blocked by incorporating one or more of several modified nucleotides.Typically these will incorporate a 3′ modification of the terminalnucleotide. Examples of these modified nucleotides are dideoxynucleotidenucleotides, 3′ amino-allyl, 3′-carbon spacers of various lengths,nucleotides incorporated in a reversed orientation (3′-3′ linkage), 3′phosphate, 3′biotin, 3′ salyl, 3′-thiol. Alternatively the terminus maycomprise nucleotides incompatible with extension be a polymerase due totheir poor substrate capability such as PNA or LNA or 2′-5′-linked DNAor 2′-O-methyl RNA.

(ii) An additional oligonucleotide (reverse complement, RC) maybesupplied that has a 3′ region cognate to the 3′ region of the IO and a5′ region cognate to the target termini as shown in FIGS. 4A-4D anddiscussed below.

The reverse complement has a 3′ region cognate to the 3′ region of theIO and binds to the outgoing template strand in this region. The 5′terminus of the RC is cognate to the target duplex proximal to the IOregion and on the outgoing strand. The 3′ region should be long enoughto bind to the outgoing strand and stable enough to induce branchmigration of its 5′ aspect into the proximal duplex. Typically this 3′region would be 10-20, preferably 10-14, bases in length.

It is important that it does not interfere significantly with thefunction of the IO and since it is cognate to this oligonucleotide itmay be preferable to include bases which are not recombinase substratessuch as 2′ modified elements, RNA or 2-O-methyl RNA or PNA or LNA.Furthermore it is helpful that this oligonucleotide is not a templatefor a polymerase. The 5′ region should be able to branch migrate intothe proximal duplex in the presence of the system constituents, in asimilar fashion to the downstream element. To this end it is preferablethat this area comprises modifications that are not a substrate forsingle stranded binding proteins.

The RC is pointing in the same direction as the downstream primer and assuch its interactions with this element are not important. The RC bindsto the opposite strand of the duplex compared with the downstreamelement and as such the downstream primer will overlap with but not becognate to the RC. Since the RC is cognate to part of the IO it may bean advantage for the IO to be blocked from extension (e.g. with spurious3′ bases or as described above) and/or have some additional bases at its3′ terminus that are not cognate to the template. This will prevent theIO from forming an amplifiable unit. The same is true for the 3′terminus of the RC and to this end the RC may be blocked at its 3′terminus to further avoid extension and may comprise some additionalbases at its 3′ terminus that are not cognate to the template.

FIGS. 3A-3D show an amplification method where a downstream element isused to protect from non-specific amplification products.

FIG. 3A: The elements of this system are shown as I-IV. A duplextemplate is shown as dotted lines. I represents an upstream primer; IIan intervening oligonucleotide (TO); III a downstream element (DE) whichis a 3′ extension of the IO and may comprise non-cognate bases at itsvery 3′ terminus. In contrast to the TO, this downstream element is nota substrate for a polymerase and may not be a substrate for therecombinase. IV represents a downstream primer.

The basis of the amplification is similar to that shown in FIGS. 2A-2Ebut in this system both of the terminal primers have a meltingtemperature that is above the ambient temperature of the system andconsequently will not amplify unless the constraints of this system aremet.

The upstream primer overlaps the IO and the downstream primer overlapsthe DE and is therefore partially cognate to this element.

All single stranded elements of the system become coated with the singlestranded binding protein GP32 although this is not necessarily the casefor the DE. UVSY which is a cofactor for UVSX also coats the IO. TheGP32 coated elements have a reduced capacity for branch migration. UVSXcompetently coats only the IO since only this element comprises asufficient length to induce the process.

FIG. 3B: The recombinase-coated IO invades the duplex melting theupstream terminus. The downstream terminus is not melted since its Tm isabove the ambient temperature and also because UVSX polymerizes ontothis area clamping the duplex together. The upstream primer binds butdoes not extend immediately because its 3′ region overlaps the IO andmust first branch migrate. It is in competition with the tetheredoutgoing template strand which outcompetes the primer for binding andthe system remains incompetent. It is also possible that the tethereddownstream terminus may branch migrate backwards closing the originalduplex after the recombinase has depolymerised but either way, thesystem will not adequately amplify.

FIG. 3C: The DE branch migrates into the downstream duplex and since theTm of the remaining downstream duplex is below the ambient temperatureit is separated. The UVSX depolymerizes and since the upstream terminusis bound to primer the duplex is completely separated.

FIG. 3D: This enables the downstream primer to bind and extend and alsogives the upstream primer the opportunity to branch migrate and extendcreating two copies of the duplex.

Notably, any primer artefact would need to comprise a binding sequencefor the DE. Since the DE is composed of elements that are not substratesfor a polymerase, this does not occur.

FIGS. 4A-4D show an amplification method utilizing a reverse complementoligonucleotide such that non-specific products cannot be formed.

FIG. 4A: The elements of this system are shown as I-IV. A duplextemplate is shown as dotted lines. I represents an upstream primer; IIan intervening oligonucleotide with non-cognate bases at its 3′terminus; III reverse complement with non-cognate bases at its 3′terminus and IV a downstream primer.

In this system both of the terminal primers have a length that is abovethe critical melting temperature of the system and consequently will notform non-specific artefacts unless the non-specific artefact isidentical to the target template.

The upstream primer overlaps the IO in the same direction. Thedownstream primer does not overlap the IO but does overlap an additionalelement, the RC. The RC is neither a polymerase nor a recombinasesubstrate and overlaps both the downstream primer and the IO. As suchthe downstream primer comprises the 3′ terminus with a sequenceidentical to a region of the 5′ area of the RC. The RC comprises a 5′sequence similar to that of the downstream primer and a 3′ sequencecomplementary to the IO.

All single stranded elements of the system except for the DE becomecoated with GP32. UVSY which is a cofactor for UVSX also coats theseelements. The coated elements have a reduced capacity for branchmigration. UVSX competently coats only the IO since only this elementcomprises a sufficient length to induce the process.

FIG. 4B: The recombinase coated IO invades the duplex melting theupstream terminus since it is below the critical temperature. Thedownstream terminus is not melted since it is above the criticaltemperature and also because UVSX polymerizes onto this area clampingthe duplex together. The upstream primer binds but does not extendimmediately because its 3′ region overlaps the IO and must first branchmigrate. It is in competition with the tethered outgoing template strandwhich outcompetes the primer for binding and the system remainsincompetent.

FIG. 4C: The UVSX de-polymerizes but the duplex strand remains partiallymelted. This enables the RC to bind to the IO and then branch migrateinto the downstream duplex.

FIG. 4D: Since the remaining duplex is below the critical temperature itis separated and falls away. This enables the downstream primer to bindand extend and also gives both the downstream and upstream primer theopportunity to branch migrate and extend creating two copies of theduplex.

Probes

Any of the methods described above may further comprise monitoring theamplification by measuring a detectable signal. The detection system maybe attached to one or more oligonucleotides that are part of theamplification system. The detection system may be fluorogenic. Asequence may be generated during amplification which is cognate to thesignal generating system.

Numerous probe based detection systems are known in the art anddescribed elsewhere, e.g. WO 2006/087574. These systems usually consistof dual labelled fluorescent oligonucleotide comprising a FRET pair of afluorophore and an acceptor moiety that maybe a fluorophore or afluorescent quencher. The probe binding sequence may be part of theamplicon downstream of the TO as shown in FIGS. 2A-2E or it may be partof the primers, TO, the RC and/or the DE. All of these elements maycomprise non-cognate bases and these may be designed such that they arecaptured by exogenous elements to localise amplified units. This iscommon to lateral flow systems.

Intercalating dyes such as Sybr green I and thiazole orange are able tosignal the general process of DNA amplification. Alternatively oradditionally a probe may be used that signals amplification of aparticular amplicon. As such probe based systems may be used tomultiplex several amplification processes in a single tube. This isachieved by utilising probes for each system with different types ofoutput. This is exemplified by different wavelengths of fluorescentemission for each probe. Multiplexing is also an important part of theprocess of including internal negative and positive experimentalcontrols.

Example of probe systems include the following:

(a) A fluorophore may be attached to the primer and this may be used asa Fret acceptor where the system includes a general intercalating dye.

(b) The fluorophore may be attached to the primer such that there is adetectable change in fluorescence when the primer is incorporated intothe amplification product. This can be achieved by placing two or morefluorophores in close proximity so that they are self quenched or groundstate quenched until incorporated into an amplification product.(c) A fluorophore and a quencher or acceptor fluorophore may beincorporated into the IO or its DE such that there is a detectablechange in fluorescence when IO is incorporated into an amplificationproduct.(d) A fluorophore acceptor/quencher (FRET) pair may be inserted into anelement of the amplification system such that they are separated by acleavable element and where the moieties of the FRET pair are separatedby the cleavable element and where the cleavable element is acted on bya duplex specific nuclease. If the element is incorporated into anamplification product then the cleavage of this element will inducecomplete separation of the FRET pair consequently enhancing fluorescenceof the system. The cleavable element maybe part of the IO or it may bepart of the primer system or an additional element added to and cognateto the amplicon of the system.

Where the cleavable element is part of the primer system then thecleavable element may be at the 5′ end of the primer binding site or atthe 3′ end of the primer binding site. If the cleavable element isplaced at the 3′ end of the moieties binding region then it may beadvantageous to place non cognate bases three prime to the cleavableelement and these bases may comprise the attachment of either thefluorophore or a quencher/acceptor. A primer with these qualities may bedesigned such that is not a part of the template amplification systembut will be included in any artefactual amplification. A primer withthese qualities can be used as a negative control for instance where ithas an area cognate to the IO at or near its 3′ terminus resulting inthe artefacts described in FIGS. 5A-5E.

The cleavage enzyme is exemplified by an RNASE-H or 8-oxoguanine or anabasic endonuclease. Typically the cleavable element will comprise RNA,8-oxoguanine or an abasic site. The RNASE-HII family of enzymesincluding that of T. kodakaraensis recognise a single RNA substrate in aDNA-RNA duplex and enables a single RNA base to be inserted into itscognate element. Additionally, the cleavage enzyme maybe a 5′-3′exonuclease such as T7 gene6 and in this case the system is protectedfrom the action of this enzyme by the application of phosphorothioateelements excepting the 5′ aspect of the oligonucleotide that containsthe fluorophore which is cleaved.

Where the cleavable element is inserted into the primer then it may beto the 5′side of the primer template binding site or 3′ to this site.Where the cleavable base is at the 3′ end of the primer binding sitethen it may be placed on the last cognate base or either side of thisbase. All bases 3′ to this element may be non-cognate to the templateand the 3′ terminus may be blocked to extension until acted on by theRNASE-H or other endonucleases. Clearly, it is important that aftercleavage either the fluorophore donor or acceptor are removed fromproximity to its partner and this is achieved by ensuring that themelting temperature at one side of the cleavage unit is below theambient temperature of the system.

(e) The fluorophore and quencher or acceptor fluorophore may beincorporated into different elements such that there is a detectablechange in fluorescence when incorporated into an amplification unit.

In a further embodiment the invention provides a kit for isothermallyamplifying a nucleic acid target molecule comprising an upstream primer,a downstream primer, a strand invasion system and an oligonucleotide,wherein the upstream and downstream primers are not substrates for thestrand invasion system during the amplification process and do notamplify the target molecule independently of the strand invasion system,wherein the oligonucleotide is a substrate for the strand invasionsystem. Each element of this kit, included preferred features, isdiscussed in detail above. For example, preferred features may includethat the strand invasion system comprises a recombinase system and/orthat the oligonucleotide comprises a downstream element at its 3′terminus.

EXAMPLES

Protocol

Reagents and Solutions:

UVSX and UVSY were purified as previously described (Timothy Formosa andBruce M. Alberts; JBC Vol. 261, 6107-6118, 1986).

RNASEHII-KOD-I was purified as previously described (Haruki M et. al. JBacteriol. 1998 December; 180(23):6207-14).

Assays were assembled from the following concentrates.

Magnesium buffer. 100 mM Tris; 100 mM Mg-Acetate; 20 mM DTT; pH 8.0

500 mM (di-Tris-) Phosphocreatine (Sigma) pH to 7.8 with ammoniumhydroxide.

200 mM DTT in H₂O

100×BSA (10 mg/ml) in H₂O

100 mM ATP-disodium salt (Jena Biosciences) in H₂O

10 mM dNTPs (Sigma D7295)

50% PEG 1000 (w/v) (Fluka) in H₂O

2 M Sucrose (Fluka) in H₂O

Creatine Kinase, Type I from Rabbit muscle (Sigma C3755) Dissolved to 10u/μl in 40% glycerol/50 mM KAc pH8

Myokinase, from Chicken muscle (Sigma M3003)

Dissolved to 9 u/μl (200×) in 40% glycerol/H₂O

Pyrophosphatase (Sigma 11643) Dissolved to 0.4 u/μl (200×) in 40%glycerol/H₂O

Sucrose Phosphorylase (Sigma S0937) Dissolved to 0.4 u/μl in 40%glycerol/H₂O

UvsX, UvsY; 100 μM in 300 mM K-Acetate; 50% glycerol.

gp32 (NEB, 10 mg/ml)

Klenow, exo⁻ (Jena Biosciences, 50 u/μl) used at final concentration of0.05 u/μl

Component Final reaction conc. Tris pH 8 10 mM Mg-Acetate 10 mM BSA**0.1 mg/ml DTT** 5 mM DMSO** 5% PEG 1000** 5% Sucrose** 150 mM ATP 2 mMdNTPs 200 μM Sybr Green** 1:100000 Oligonucleotides As show in examplesgp32 0.5 uM Phosphocreatine 75 mM (diTRIS) pH to 7.8 With KOH CreatineKinase 1 uM Myokinase** 1 uM Pyrophosphatase** 1 uM UvsY 1.5 uM UvsX 1.5uM Sucrose 1 uM phosphorylase** Klenow 0.1 uM Template DNA As shown inexamples **= components found to optimise but are not essential foramplification.

Test template was added to a mixture of the reaction components exceptfor UVSX and klenow. The reaction components were incubated with testsample for 5 minutes at the working temperature (40° C.) and the UVSXand klenow were added. Total sample volumes were 20 ul placed into a lowvolume 384 well micro-titre plates. Fluorescence was assessed on aBMG-fluostar-II. Fluorescence was monitored at one minute intervals byexciting at 480 nm and reading emission at 520 nm for Sybr greenfluorescence (unless otherwise stated).

Example 1 Two-Primer System, No Intermediate Oligonucleotide (Prior ArtSystem)

The protocol used is the same as that described above unless otherwisestated. The oligonucleotide constituents comprised two primers at afinal concentration of 150 nM together with Template A. Template/primerconfigurations are shown in FIG. 6A. Template A concentrations were 1 nMunless stated and amplification was followed by Sybr green fluorescence.

The results are shown in FIG. 6B. In this two primer system,amplification occurred where the primer length was equal to or in excessof 32 bases (U32+D32; U35+D35 and U40+D40). No amplification occurredwith the 23 and 20 base primers (U23+D23 and U20+D20). Primer dimerartefacts occurred as shown for the 40 base primers (no template). Theseartefacts generally emerged at the same time as template concentrationsof 10 pM and this limited the sensitivity of the technique to thislevel.

Example 2—Three-Primer System (Using Two Primers and a Non-ExtendibleIntermediate Oligonucleotide)

The protocol used is the same as that described above unless otherwisestated. Template/oligonucleotide/primer configurations are shown in FIG.7A. Primers were U16 and D16 used at a concentration of 200 nM each.Intermediate oligonucleotide concentrations were 150 nM and templateconcentrations were 100 fM. The signal was produced by Sybr greenfluorescence.

FIG. 7B demonstrates amplification of the three oligonucleotide systemconfigured as shown in FIG. 7A using upstream and downstream primers U16and D16 respectively. Amplification is achieved with primers of 16 basesin length and is dependent on the non-extendible oligonucleotide (TO).The system is less prone to artefacts. The primers of 16BP are able toamplify if the intermediate oligo is cognate (IO1+primers+Template1;IO2+primers+Template2)). Primers will not amplify alone (primers only);neither will intermediate amplify alone (no primers). Artefacts canoccur in the absence of template in some systems limiting thesensitivity to between 1 and 10 fM giving a sensitivity of one thousandtimes greater than a two primer system.

Example 3—Primers in a Tripartite System Must be Below 20 Bases inLength Under the Conditions Used (Dependent on Melt Temperature of thePrimer and Ambient Temperature and Denaturation Agents)

The protocol used is the same as that described above unless otherwisestated. Primers were of various lengths were used at a concentration of200 nM each. Intermediate oligonucleotide (IO1) was used at 150 nM andtemplate concentration was 100 fM. The signal was produced by Sybr greenfluorescence.

The results are shown in FIG. 8. Primers of 12, 14 and 16 basesamplified efficiently. The primer set of 18 bases amplified lessefficiently and the 20 base primer set did not amplify and thereforewould not form artefacts.

Example 4—Artefacts can be Abrogated Using Methylated DownstreamElements in the Intermediate Oligonucleotide

The protocol used is the same as that described above unless otherwisestated. Template/oligonucleotide/primer configurations are shown in FIG.9A. Primers were used at a concentration of 300 nM each. Intermediateoligonucleotide (101) was used at 150 nM and template concentration was10 pM. The signal was produced by Sybr green fluorescence.

In the three primer system longer primers (20BP) are unable to amplify(IO1+U20+D20 and IO1met+U20+D20) since the regions upstream anddownstream of the invading intermediate oligonucleotide are too long toseparate and allow the primers to bind. Furthermore after theintermediate oligonucleotide binding event is complete and therecombinase depolymerises (falls off the oligonucleotide), the duplex,which is not fully separated tends to close again by branch migration.

If the downstream primer is long and the upstream primer is short thenamplification is slow (IO1+U16+D20) but visible. This is because theupstream region is separated when the IO invades allowing the upstreamprimer to bind but it has to compete with the partially tethered duplexduring extension.

If the upstream primer is long but overlaps the intermediate then someslow amplification is also observed with a long downstream primer(IO1+U20over+D20). This is because, although the primer is long theregion of the amplified template upstream of the 10 remains short (seeFIG. 9A).

If the downstream primer is long and a methylated downstream element isincorporated into the intermediate (IO1meth+U16+D20 andIO1meth+U20over+D20) then the amplification rate is increased to thatsimilar to a short downstream primer. This is because the methylatedregion of the 10 branch migrates into the downstream region of theduplex shortening the remaining duplex, separating the duplex strandallowing primer to bind and freeing extension of the upstream primerfrom competition with a partially tethered template. The methylatedelement must be cognate and therefore able to branch migrate sinceIO1Met2+U20over+D20 does not show accelerated amplification. This is thecase even where the upstream primer is long if it overlaps theintermediate (IO1meth+U20over+D20).

Despite the rapid amplification using long primers and the methylatedintermediate, if the long upstream primer does not overlap theintermediate then there are is no amplification and no artefacts(IO1meth+U20+D20). It is likely that this is due to the concept thatwhere the upstream region remains intact the amplification must beinitiated by the downstream primer. The region downstream of theintermediate tends to be covered in recombinase until the recombinasedepolymerises and consequently cannot bind primer until the recombinasehas depolymerised by which time the duplex has closed by branchmigration. These observations demonstrate that rapid amplification usinga methylated intermediate oligonucleotide and long primers is possibleand relies on a cognate methylated region of the intermediate. Since thecognate region of the methylated moieties are not substrates for apolymerase, artefacts are not possible.

Example 5—the Three Oligonucleotide System can Amplify from BiologicallyDerived DNA

The protocol used is the same as that described above unless otherwisestated and the oligonucleotide are as shown in the legend and configuredas shown in FIG. 9A. Primers were used at a concentration of 300 nMeach. Intermediate oligonucleotide (IO1) was used at 150 nM and templateconcentration was 1 fM. Template1 with an additional ALU1

sensitive agct sequence immediately upstream of the oligonucleotide wasinserted into the plasmid vector PXero-2 by IDT-DNA (San-Diego). Thesignal was produced by Sybr green fluorescence. Plasmid was cleaved withALU1 using 0.1 ug of plasmid in 100 ul (1 nM) and digestion for 30 minsat 37° C. with 5 units of ALU1 (New England Biolabs) in NEB buffer 4.The plasmid template was subsequently diluted in water as described inthe standard operating procedure.

Amplification of this template1 inserted into the plasmid was comparedwith that of the synthetic template and also with the plasmid cleavedimmediately upstream of the template sequence. As discussed above(example 4), the region of template upstream of the intermediateoligonucleotide needs to be short for efficient amplification. In abiological system the target template is usually part of a long sequenceof DNA and the duplex upstream of the intermediate oligonucleotide maybe longer than that desired for efficient amplification. This couldeffect the first cycle of amplification in such systems unless thetemplate is heated prior to amplification rendering the template singlestranded. The importance of this issue was assessed using plasmid DNA.

The results are shown in FIG. 10. There was a delay of several minutesfor amplification of the plasmid and a smaller delay where the plasmidwas cleaved upstream of the template. It may be that negative supercoiling of the plasmid facilitated the first round of amplificationhowever a plasmid cleaved downstream of the template also producedamplification after a similar delay. Alternatively occasional breathingof the duplex may have enabled amplification after a delay.Amplification of the no template control was seen but this was after asingle molecule of the template would have been detected. It is likelythat the methylated portion of IO1 was copied at a very slow rate by thepolymerase eventually forming an artefact. There was only a singlemethylated base between overlap of the primer terminus with themethylated region of the intermediate and the DNA portion of theintermediate which may have enabled eventual read-through in this area.In subsequent experiments the number of bases between the primer and theDNA portion of the intermediate were increased to avoid all artefactualamplification.

Example 6—the Sensitivity of the System Using a Methylate Intermediatecan be at the Level of a Single Molecule

The protocol used is the same as that described above unless otherwisestated. The oligonucleotides used were U20over, template1, IO1methextraand D20back. This configuration was used to avoid any artefactualamplification events since the IOmethextra comprises additional2′methylated RNA bases compared with IO1meth decreasing further theopportunity of the downstream primer to copy through the additional2′-methylated bases to the point that this becomes implausible. Theoligonucleotide configuration is shown in FIG. 9A. The signal wasproduced by Sybr green fluorescence.

The results are shown in FIGS. 11A and 11B. In FIG. 11A the intermediatewas used at 75 nM whereas in FIG. 11B the intermediate was used at 150nM. Primer concentrations were 200 nM. In FIG. 11A 1 million, 1000 andone hundred molecules were added. In FIG. 11B the assay comprised theaddition of 10, 5, 0.5, 0.05 and 0 molecules of template 1 to each testsuch that 0.5 molecules had a ½ chance of containing a single molecule.Three samples of each concentration were prepared and the results areshown. All samples with 10 and five molecules amplified. One of thethree samples that had a ½ chance of containing a molecule amplified(the sample with amplification delayed). No other samples amplified.

Example 7—Crowding Agents can Improve the Kinetics of the System

The protocol used is the same as that described in Example 1 unlessotherwise stated. The oligonucleotides are as shown in the legend andconfigured as shown in FIG. 9A. Primers were U16 and D16 used at aconcentration of 200 nM each. Intermediate oligonucleotide (IO1)concentrations were 150 nM and template-1 concentration was 100 pM. Thesignal was produced by Sybr green fluorescence.

The results are shown in FIG. 12. The system was viable without crowdingagents but amplified more efficiently in the presence of different typesof PEG or albumin as previously reported (Reddy M K et. Al. MethodsEnzymol. 1995; 262:466-76; Lavery P et. Al. JBC 1992, 26713, 9307-9314;WO2008/035205).

Example 8—the Amplification can be Interrogated by Probes Instead ofSybr Green in Order to Multiplex the Reaction or for the Purpose ofIncorporation of Positive and Negative Controls

The protocol used is the same as that described in Example 1 unlessotherwise stated. The oligonucleotides used were U20over (200 nM),template1, IO1meth (75 nM), and a mixture of D20 and D20probe at theconcentrations of 100 nM each. The concentration of template was 100 fM.The oligonucleotide configuration is shown in FIG. 9A. RNASEH from T.kodakaraensis was added at a final concentration of 1 nM together withthe other components. The system was excited and read at 480/520 nm toassess amplification or 540/600 nm to interrogate probe cleavage. Theprobe was incorporated as part of the downstream primer system. Theprimer comprised a template cognate region, an RNA base and a blockednon cognate region at its 3′ terminus. The RNA base was cleaved byRNASEHII when the primer bound to template allowing the primer toextend. Since the primer comprised a quencher and fluorophore eitherside of the RNA base, they became separated on cleavage of the RNA baseproducing a signal. FIG. 13 shows both the signal produced by Sybr greenand the signal induced by the probe.

A probe primer designed to assess the presence of a template could beused together with an additional probe primer incorporating analternative fluorophore. Such a system can be configured for the purposeof positive and negative controls where a control template is added at aknown concentration as part of the system. Alternatively, and in theevent that the system could induce artefactual amplification, then aprobe primer that induces earlier artefactual amplification could beadded and where a signal is produced by the amplification of such aprobe then the test would be terminated. This is exemplified by the useof D20control probe and D20back in FIG. 9a where D20control will induceearlier artifactual amplification due to its closer proximity to the DNAbases of the intermediate.

Sequences

U40 (SEQ ID NO: 1) GTTACGATTGTCCTAATGGAGAGTGAGTTGTGATGATGTC U35(SEQ ID NO: 2) GATTGTCCTAATGGAGAGTGAGTTGTGATGATGTC U32 (SEQ ID NO: 3)TGTCCTAATGGAGAGTGAGTTGTGATGATGTC U23-overlap (SEQ ID NO: 4)GAGTTGTGATGATGTCATTCGCA U23 (SEQ ID NO: 5) CGAGAGTGAGTTGTGATGATGTC U20(SEQ ID NO: 6) GAGTGAGTTGTGATGATGTC U18 (SEQ ID NO: 7)GTGAGTTGTGATGATGTC U15 (SEQ ID NO: 8) AGTTGTGATGATGTC U12 (SEQ ID NO: 9)TGTGATGATGTC D40 (SEQ ID NO: 10)TCTGGCATGTTACAAGGTCAAGATGAACCAACCACTTATA D35 (SEQ ID NO: 11)CATGTTACAAGGTCAAGATGAACCAACCACTTATA D32 (SEQ ID NO: 12)GTTACAAGGTCAAGATGAACCAACCACTTATA D23 (SEQ ID NO: 13)TCAAGATGAACCAACCACTTATA D20 (SEQ ID NO: 14) AGATGAACCAACCACTTATA D18(SEQ ID NO: 15) ATGAACCAACCACTTATA D16 (SEQ ID NO: 16) GAACCAACCACTTATAD14 (SEQ ID NO: 17) ACCAACCACTTATA D12 (SEQ ID NO: 18) CAACCACTTATAD20back (SEQ ID NO: 19) GGTCAAGATGAACCAACCAC X =blocked base comprising 3′ amino-6carbon-spacer X = 2′-O-methyl RNA X =RNA base TQ is a T attached to a BHQ2TF is a T attached to Tetramethylrhodamine (TAMRA) IO1 (SEQ ID NO: 20)TGAGCATAGACGGCATTCGCAGATCCAGTCAGCAGTTCTTCTCACTCTTCA A IO2(SEQ ID NO: 21) GAGGCTAAGGAATACACGCAAAGGCGGCTTGGTGTTCTTTCAGTTCTTCAAIO1met (SEQ ID NO: 22)TGAGCATAGACGGCATTCGCAGATCCAGTCAGCAGTTCTTCTCACTCTTCA AGTATA GIO1met extra (SEQ ID NO: 23)TGAGCATAGACGGCATTCGCAGATCCAGTCAGCAGTTCTTCTCACTCTTCA AGTATAAGTGG AIO1met2 (SEQ ID NO: 24)TGAGCATAGACGGCATTCGCAGATCCAGTCAGCAGTTCTTCTCACTCTTCA ATTCTA G Template A(SEQ ID NO: 25) GTTACGATTGTCCTAATGGAGAGTGAGTTGTGATGATGTCCTGTATAAGTGGTTGGTTCATCTTGACCTTGTAACATGCCAG Template1 (SEQ ID NO: 26)GTTACGATTGTCCTAATGGAGAGTGAGTTGTGATGATGTCATTCGCAGATCCAGTCAGCAGTTCTTCTCACTCTTCAAGTATAAGTGGTTGGTTCATCTTGA CCTTGTAACATGCCAGTemplate 2 (SEQ ID NO: 27)GTTACGATTGTCCTAATGGAGAGTGAGTTGTGATGATGTCACACGCAAAGGCGGCTTGGTGTTCTTTCAGTTCTTCAAGTATAAGTGGTTGGTTCATCTTGA CCTTGTAACATGCCAGD20probe (SEQ ID NO: 28) AGATGAACCAACCAC(TQ)TATATTT(TF)TTT D20probe2(SEQ ID NO: 29) T(TF)TTTTTAGA(TQ)GAACCAACCACTTATA

The invention claimed is:
 1. A method of ATP regeneration in arecombinase enzyme reaction comprising providing to the recombinaseenzyme reaction phosphocreatine, creatine kinase, sucrose, and sucrosephosphorylase wherein sucrose and sucrose phosphorylase reacts withaccumulated orthophosphate.
 2. The method of claim 1, wherein therecombinase is UvsX.
 3. The method of claim 1, wherein a concentrationof 40-100 mM phosphocreatine is used.
 4. The method of claim 1, furthercomprising providing to the recombinase enzyme reaction a myokinase,wherein AMP is converted to ADP with the myokinase.
 5. The method ofclaim 1, wherein the reaction further comprises pyrophosphatase toconvert pyrophosphate to orthophosphate.
 6. The method of claim 1,wherein ATP regeneration is used in isothermal DNA amplification.
 7. Acomposition for ATP regeneration comprising a recombinase, enzymes whichenzymatically convert AMP to ADP or ATP, sucrose and sucrosephosphorylase, and phosphocreatine and creatine kinase.
 8. A compositionof claim 7, comprising myokinase.
 9. A composition of claim 7,comprising pyrophosphatase.
 10. A composition of claim 7, wherein therecombinase is UvsX.
 11. The method of claim 1, further comprisingproviding to the recombinase enzyme reaction one or more energy sourcesselected from phospho-phenyl-pyruvate and pyruvate kinase; ATP;myokinase; pyrophosphatase; and any combination of two or more of theforegoing.
 12. The composition of claim 7, comprisingphospho-phenyl-pyruvate and pyruvate kinase.
 13. The composition ofclaim 7, comprising ATP.